This project seeks to understand the metabolic origins of skeletal muscle insulin resistance, a physiological abnormality common to obesity, diabetes and aging. In recent studies that applied targeted metabolomics we discovered that the early stages of diet-induced weight gain and glucose intolerance are accompanied by increased fat oxidation and intramuscular accumulation of mitochondrial-derived acylcarnitine metabolites, byproducts of incomplete substrate catabolism. Likewise, the addition of branched chain amino acids (BCAA) to a high fat diet exacerbated insulin resistance while provoking a further increase in muscle levels of both lipid- and amino acid-derived acylcarnitines. These metabolomic signatures suggest that the mechanisms underlying diet-induced insulin resistance might be directly related to carbon load within the mitochondrial compartment. Thus, the overarching goal of this project is to test the hypothesis that excessive mitochondrial lipid and BCAA catabolism plays a central role in triggering mitochondrial stress, insulin resistance and eventual metabolic failure during the pathological progression of diet-induced obesity. Our working model predicts that acylcarnitines accumulation in the obese state reflects a mitochondrial environment that is conducive to hyperacetylation of mitochondrial proteins and increased generation of reactive oxygen species. These hypotheses will be tested by combining state-of-the-art metabolomics and metabolic flux analyses with genetically modified mouse models harboring targeted manipulations in fat oxidation and acylcarnitines production. This project is germane to current antiobesity and antidiabetic drug development efforts aimed at increasing skeletal muscle fat oxidation, and could lead to paradigm shifting insights into the interplay between mitochondrial function and insulin action in muscle.